Multicentre study to determine the Etest epidemiological cut-off values of antifungal drugs in Candida spp. and Aspergillus fumigatus species complex
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(2) rip. t. Multicentre study to determine the Etest epidemiological cutoff values of antifungal drugs in Candida spp. and Aspergillus fumigatus species complex. M an us c. M. Salse' 1, J.-P. Gangneux 2, S. Cassaing 3, L. Delhaes 4, A. Fekkar 5, D. Dupont 6, F. Botterel 7, D. Costa 8, N. Bourgeois 9, B. Bouteille 10, S. Houze' 11, E. Dannaoui 12, H. Guegan 2, E. Charpentier 3, F. Persat 6, L. Favennec 8, L. Lachaud 9, M. Sasso 1, *. 1). Service de Parasitologie-Mycologie, CHU Nîmes, Universit'e Montpellier, Nîmes, France CHU de Rennes, Institut de Recherche en Sant'e Environnement et Travail, UMR U1085 Inserm-Universit'e Rennes 1, Rennes, France Service de Parasitologie-Mycologie, CHU Toulouse, Universit'e Paul Sabatier, Toulouse, France 4) Service de Parasitologie-Mycologie, CHU Bordeaux, Bordeaux, France 5) AP-HP, Groupe Hospitalier Piti'e-Salp^ etri'ere, Service de Parasitologie-Mycologie, F-75013, Paris, France 6) Hospices Civils de Lyon, Institut des Agents Infectieux, Parasitologie-Mycologie M'edicale, Universit'e Lyon 1, Lyon, France 7) Unit'e de Parasitologie-Mycologie, CHU Henri Mondor, APHP, Paris, France 8) Laboratoire de Parasitologie-Mycologie, CHU Rouen, Universit'e de Normandie, EA 7510, Rouen, France 9) Service de Parasitologie-Mycologie, CHU Montpellier, Universit'e de Montpellier, UMR Mivegec, Montpellier, France 10) Service de Parasitologie-Mycologie, CHU Limoges, Limoges, France 11) APHP Bichat, Laboratoire de Parasitologie-Mycologie, Paris, France 2) 3). 12). ^pital Europ'een Georges Pompidou, Laboratoire de Parasitologie-Mycologie, D'epartement de Universit'e Paris-Descartes, Facult'e de M'edecine, APHP, Ho Microbiologie, Paris, France. A b s t r a c t. Ac. ce. pt. ed. Objectives: To determine the Etest-based epidemiological cut-off values (ECVs) for antifungal agents against the most frequent yeast and Aspergillus fumigatus species isolated in 12 French hospitals. Methods: For each antifungal agent, the Etest MICs in yeast and A. fumigatus isolates from 12 French laboratories were retrospectively collected from 2004 to 2018. The ECVs were then calculated using the iterative statistical method with a 97.5% cut-off. Results: Forty-eight Etest ECVs were determined for amphotericin B, caspofungin, micafungin, anidula- fungin, fluconazole, voriconazole, posaconazole and itraconazole, after pooling and analysing the MICs of 9654 Candida albicans, 2939 Candida glabrata SC, 1458 Candida parapsilosis SC, 1148 Candida tropicalis, 575 Candida krusei, 518 Candida kefyr, 241 Candida lusitaniae, 131 Candida guilliermondii and 1526 Aspergillus fumigatus species complex isolates. These ECVs were 100% c oncordant (identical or within one two-fold dilution) with the previously reported Etest-based ECVs (when available), and they were concordant in 76.1% of cases with the Clinical and Laboratory Standards Institute ECVs and in 81.6% of cases with the European Committee on Antimicrobial Susceptibility Testing ECVs. Conclusions: On the basis of these and other previous results, we recommend the determination of method-dependent ECVs. Etest ECVs should not be used instead of breakpoints, but may be useful to identify non-wild-type isolates with potential resistance to antifungal agents, and to indicate that an isolate may not respond as expected to the standard treatment.. ' Montpellier, Nîmes, France. * Corresponding author. M. Sasso, Service de Parasitologie-Mycologie, CHU Nîmes, Universite E-mail address: milene.sasso@chu-nimes.fr (M. Sasso)..
(3) Introduction. MIC data collection. ed. Material and methods. M an us c. rip. t. The prevalence of invasive infections due to fungal pathogens continues to increase in immunocompromised individuals. In France, candidiasis represents the primary cause (49%) of invasive fungal diseases, of which 56% are due to Candida albicans, 18.6% to Candida glabrata, 11.5% to Candida parapsilosis, and 9.3% to Candida tropicalis. Aspergillosis is in third position (16% of all invasive fungal diseases) [1]. To manage these invasive fungal diseases, three classes of antifungal agents (polyenes, echinocandins and azoles) are recommended as initial or salvage therapy [2,3]. The Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) have established standard procedures for testing the susceptibility of fungi to anti- fungal agents, and have proposed species-specific breakpoints for interpreting the MICs of some antifungal agents against the most prevalent Candida spp. [4e6]. Species-specific breakpoints predict the likelihood of clinical response to antimicrobial therapy. How- ever, there are many pathogeneantifungal agent combinations for which clinical breakpoints are not available, because of insufficient data to correlate the clinical outcomes with the in vitro results. When only MIC data are available, epidemiological cut-off values (ECVs or ECOFF) should be considered. ECVs are defined as the highest susceptibility end point of the MIC distribution for the wild- type (WT) population. A MIC value higher than the ECV suggests that the isolate may have developed resistance to that agent, so an alternative compound should be considered [7,8]. ECVs are dependent on the in vitro sensitivity testing method used to generate the MIC values. Although ECVs for several organismeantifungal agent combinations have been published [9e19], few data are available on ECVs based on the MIC distribu- tions obtained with commercial methods. As the gradient agar diffusion-based Etest assay (bioMe'rieux, Marcy l’Etoile, France) is often used in the clinical routine [10,11,13], we collected retro- spectively the MICs for yeast and Aspergillus fumigatus species complex (SC) isolates obtained with the Etest method at 12 French mycological laboratories to determine the ECVs of the main agents used in the clinical practice, in order to harmonize the interpreta- tion of the Etest MIC results.. pt. The MICs for different antifungal agents tested on cultured pa- tient isolates with the Etest from 2004 to 2018 were collected retrospectively from the laboratories of 12 French university hos- pitals (CHU de Nîmes, CHU de Rennes, CHU de Toulouse, CHU de Bordeaux, Hospices Civils de Lyon, CHU de Rouen, CHU de 'trie're, APHP Ho^ pital BichatMont- pellier, CHU de Limoges, APHP Ho^ pital universitaire Pitie'-Sal- pe ^ ' ^ Claude Bernard, APHP Ho pital Europeen Georges Pompidou and APHP Ho pital Henri Mondor).. ce. Species identification and antifungal susceptibility testing. Ac. The tested isolates were from routine specimen cultures (blood cultures, sterile sites and other sites, such as bronchoalveolar lavage, sputum) that required antifungal susceptibility testing for therapeutic management. Fungi were identified using different methods: phenotypic features in chromogenic medium, microscopic morphology, VITEK®2 YST system (bioMe'rieux), API® ID32C (bioMe'rieux) or mass spectrometry (Bruker Microflex LT™ system, BrukerDaltonik, Bremen, Germany, or VITEK® MS, bioMe'rieux). The Etest-based MIC values for the following species could be collected: C. albicans, C. glabrata SC, C. parapsilosis SC, C. tropicalis, Candida krusei, Candida kefyr, Candida lusitaniae, Candida guillier- mondii and A. fumigatus SC. Each laboratory determined the isolate susceptibility to different antifungal drugs (amphotericin B, anidulafungin, caspofungin, micafungin, 5-fluorocytosine, fluconazole, itracona- zole, posaconazole and voriconazole) using the Etest gradient diffusion method, according to the manufacturer's instructions [20]. Etest MICs were determined by visual observation at 48 h of growth for yeasts, and between 16 and 72 h, depending on their growth, for A. fumigatus SC. During the study period, all contrib- uting laboratories tested the quality control reference strains. The MICs obtained for these strains were all within the expected reference ranges. All Etest MIC readings were performed by quali- fied operators. In 2018, in parallel with data collection, to test the inter-operator variability among centres, each reader determined the MICs based on visual inspection of 16 photographs of Etest assays..
(4) 4. No significant difference between the determined MICs was observed.. ECV determination. M an us c. rip. t. To ensure that robust and comparable data were included for the ECV estimation, the following basic requirements and criteria needed to be fulfilled [9]. (i) MICs were converted into the standard two-fold dilution scale based on dilution factor values that are powers of 2 (at the upper dilution). All data were collected in the form of number of isolates with different MIC values on the power of 2 scale. The MIC mode was the MIC value with the highest number of representative isolates. (ii) If the MIC mode of a distri- bution was at the lowest tested concentration, or if the distribution appeared truncated before or after the MIC mode, that distribution was excluded. (iii) Distributions with aberrant MIC modes were excluded: for example, when the MIC mode of the distribution was two-fold or more than two-fold dilutions above or below the most frequent WT mode. (iv) MIC data were not pooled if there was no obvious common mode among the range of distributions (for example bimodal distributions). (v) If one of the participating lab- oratories contributed >50% of the values to the pooled data, the MIC data were normalized to reduce this bias in the estimate. (vi) MIC data were pooled only if generated by at least three independent laboratories. (vii) For each specieseantifungal agent combination, a minimum of 100 MIC values were required after data pooling. The ECVs from pooled data were estimated using the iterative statistical method (ISM) described by Turnidge et al. [7] and implemented in a MICROSOFT EXCEL® ECOFFINDER workbook (https:// www.clsi.org/meetings/microbiology/ecoffinder/). This method selects the log normal distribution for subsequent modelling. From the real MIC distribution (power of 2 scale), the ISM attempts to fit iteratively the observed WT counts in a log normal distribution and creates a range of possible ECVs. This fitted log-normal distribution is a probability distribution of the WT population. Each resulting Etest ECV corresponded to the MIC that captured 97.5% of the modeled WT population, and represented the probability for an isolate to be a WT isolate if its MIC was lower or equal to the ECV value.. ed. Ethics. Results. pt. This study included only data from fungal isolates. The opinion of an Institutional Review Board was not required because human participants were not involved.. Ac. ce. Analysis of the data allowed determination of 48 ECVs for nine yeast and one mould species (total number of included isolates: 9654 C. albicans, 2939 C. glabrata SC, 1458 C. parapsilosis SC, 1148 C. tropicalis, 575 C. krusei, 518 C. kefyr, 241 C. lusitaniae, 131 C. guilliermondii and 1526 A. fumigatus SC). Depending on the species and antifungal agent, MIC data from 3 to 12 laboratories were pooled. The MIC distributions for each speciesemolecule combination are presented in Table 1. The Etest-based ECVs of amphotericin B and echinocandins for Candida spp. are presented in Table 2. ECVs were determined for the five main species (C. albicans, C. glabrata, C. parapsilosis SC, C. tropicalis and C. krusei) using MIC data on 117 to 6062 isolates, according to the specieseantifungal agent combination. Enough data (>100 isolates) were collected to determine new ECVs for less prevalent species, such as C. kefyr, C. guilliermondii and C. lusitaniae. The MICs determined for Candida spp. and azoles (Table 3) allowed the calculation of 15 ECVs. It was not possible to estimate the Etest-based ECVs for C. glabrata and azoles because of the aberrant MIC distribution observed with the Etest method (double peak). Moreover, the ECV for the C. kruseiefluconazole combination was not determined because of its innate resistance to this drug. To calculate the ECVs for A. fumigatus SC (Table 4), data on 361 to 1027 isolates were used, depending on the specieseantifungal agent combination. Two new ECVs were determined for echino- candins. The ECVs for A. fumigatus SC and amphotericin B and azoles were similar to those previously reported..
(5) Ac. ce. pt. ed. M an us c. rip. t. Overall, the ECV results were identical to (17/32 ECVs) or within one two-fold dilution of (15/32 ECVs) the previously reported Etest- based ECVs (Tables 2e4). They were comparable to the CLSI ECVs in 76.1% (similar or within one two-fold dilution: 35/46 ECVs) and absolutely identical in 39.1% of cases (18/46 ECVs). They were comparable to the EUCAST ECVs in 81.2% of cases (similar or within one two-fold dilution: 26/32 ECVs) and strictly identical in 28% (9/ 32 ECVs)..
(6) Candida tropicalis. Candida krusei. Candida kefyr. Candida lusitaniae. Candida guilliermondii. a. 0.002. 0.004. 0.008. 0.016. 0.032. 0.064. 0.12. 6062 5783 3752 1593 9654 6020 1005 2272 2292 1494 513 2939 2304 455 981 1091 839 241 1458 1150 787 787 504 213 1148 886 152 534 565 259 117 414 575 353 418 236 518 357 170 149 241 168 131 120 111 115 1027 806 361 1526 961 989. 11 12 11 5 12 12 6 11 11 11 5 11 11 7 10 10 11 3 12 12 10 10 11 5 12 12 4 9 10 5 3 11 10 9 8 7 11 9 9 7 10 8 6 5 5 5 9 5 5 7 8 7. 6 0 0 3 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0. 8 5 6 560 0 327 5 0 0 0 0 0 4 0 1 0 0 0 0 31 0 1 0 2 0 2 1 0 0 1 2 0 0 0 0 0 0 20 0 0 0 20 0 0 0 0 1 2 14 1 2 0. 0 14 59 674 0 1551 13 0 3 2 8 0 8 0 0 0 2 0 0 111 0 1 0 7 0 5 0 0 0 0 0 0 0 1 0 0 0 63 0 0 0 39 0 0 0 0 0 12 90 1 2 0. 8 125 855 289 0 2161 151 0 0 98 67 0 13 1 1 1 1 0 0 264 1 2 1 50 0 16 7 1 0 1 0 0 0 0 0 0 0 141 1 0 0 67 1 0 0 2 0 65 165 1 4 0. 18 615 1909 38 5 1272 428 4 16 988 353 1 16 3 5 0 2 0 4 285 1 18 61 123 1 106 32 1 0 1 27 0 2 2 8 2 1 95 0 3 2 23 12 0 0 25 1 193 75 1 20 1. 92 1572 822 9 59 434 288 11 44 359 61 0 57 2 17 2 1 0 8 236 3 59 338 24 1 211 45 4 1 3 43 0 5 8 49 30 4 29 7 5 9 3 14 0 0 26 6 251 14 13 68 4. 297 1891 62 7 519 141 72 31 312 7 8 2 120 5 28 8 0 1 22 118 29 208 96 4 3 308 34 6 3 7 37 0 20 26 113 126 39 4 7 14 21 3 28 2 0 22 9 191 1 50 367 10. 1079 1220 13 3 2501 57 25 93 1121 5 4 5 326 14 53 28 4 1 111 47 49 300 4 2 15 165 21 18 16 106 5 0 79 45 212 73 141 2 28 39 44 5 25 10 0 6 63 75 1 662 391 39. The MIC mode (most frequent value) for each distribution is in bold type.. 0.25. 0.5. 1. 2. 4. 8. 16. 32. 64. 128. 256. >256. 2803 294 9 6 3880 36 8 288 688 6 4 9 409 24 155 203 24 4 337 18 146 163 1 0 82 31 9 40 139 137 3 0 183 90 33 5 197 2 70 59 83 3 34 34 1 5 225 16 1 622 75 190. 1569 34 9 0 1908 13 7 995 66 10 0 20 423 49 390 395 102 16 445 11 295 23 0 0 301 16 2 144 303 2 0 3 212 161 3 0 114 1 47 24 54 3 15 41 10 7 505 1 0 109 17 474. 159 5 6 0 488 6 6 712 19 3 3 28 266 56 271 304 388 60 288 8 222 6 0 0 409 8 1 207 81 1 0 1 49 16 0 0 15 0 5 4 13 1 1 23 26 6 178 0 0 29 3 199. 19 1 1 1 147 4 1 113 4 9 1 97 145 63 48 119 234 80 124 8 36 3 3 1 229 3 0 96 18 0 0 1 14 4 0 0 4 0 5 1 2 0 1 4 31 3 27 0 0 18 6 35. 1 2 0 2 48 0 1 16 3 3 2 204 117 46 9 22 73 43 40 9 5 2 0 0 52 1 0 15 4 0 0 0 7 0 0 0 0 0 0 0 2 0 0 2 9 0 7 0 0 10 1 4. 0 2 0 0 21 0 0 2 4 3 1 375 95 35 1 4 5 12 24 1 0 0 0 0 22 2 0 1 0 0 0 1 2 0 0 0 1 0 0 0 3 0 0 1 10 1 3 0 0 4 0 8. 1 1 0 1 20 0 0 4 1 0 1 722 72 14 0 1 3 3 19 0 0 0 0 0 9 2 0 0 0 0 0 21 1 0 0 0 0 0 0 0 2 0 0 0 4 0 1 0 0 2 1 6. 0 1 1 0 12 8 0 0 1 1 0 557 61 58 0 0 0 4 11 0 0 0 0 0 5 3 0 1 0 0 0 65 0 0 0 0 0 0 0 0 3 0 0 0 5 3 1 0 0 0 0 4. 2 1 0 0 7 5 0 3 6 0 0 213 172 85 2 4 0 17 5 3 0 1 0 0 4 7 0 0 0 0 0 81 1 0 0 0 0 0 0 0 1 0 0 3 3 3 0 0 0 3 4 15. 0 0 0 0 1 0 0 0 0 0 0 91 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 23 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0. 0 0 0 0 10 0 0 0 0 0 0 100 0 0 0 0 0 0 3 0 0 0 0 0 1 0 0 0 0 0 0 47 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0. 0 0 0 0 28 0 0 0 4 0 0 515 0 0 0 0 0 0 17 0 0 0 0 0 13 0 0 0 0 0 0 171 0 0 0 0 0 0 0 0 2 0 0 0 10 6 0 0 0 0 0 0 3. Ac. Aspergillus fumigatus SC. 0.001. an us. Candida parapsilosis SC. No. of isolates with MIC (mg/L) ofa:. M. Candida glabrata SC. Amphotericin B Caspofungin Micafungin Anidulafungin Fluconazole Voriconazole Posaconazole Amphotericin B Caspofungin Micafungin Anidulafungin Fluconazole Voriconazole Posaconazole Amphotericin B Caspofungin Micafungin Anidulafungin Fluconazole Voriconazole Amphotericin B Caspofungin Micafungin Anidulafungin Fluconazole Voriconazole Posaconazole Amphotericin B Caspofungin Micafungin Anidulafungin Fluconazole Voriconazole Amphotericin B Caspofungin Micafungin Fluconazole Voriconazole Amphotericin B Caspofungin Fluconazole Voriconazole Amphotericin B Caspofungin Fluconazole Voriconazole Amphotericin B Caspofungin Micafungin Voriconazole Posaconazole Itraconazole. From number of laboratories. ed. Candida albicans. Tested isolates (n). pt. Antifungal drug. ce. Species. cr ip t. M. Sals'e et al. / Clinical Microbiology and Infection xxx (xxxx) xxx Table 1 Pooled MIC distributions of Candida species and Aspergillus fumigatus SC.
(7) 5. Discussion. M an us c. rip. t. The Etest method is widely used in the clinic, and MIC inter- pretation is based on the CLSI breakpoints that are, however, available only for some specieseantifungal drug combinations [21]. In their absence, the method-dependent ECVs allow identification of non-WT isolates that might be resistant to an antifungal agent. ECVs for some specieseantifungal agent combinations have been established using both reference methodsdCLSI [9,12,15e19] and EUCAST [9,10,14,22,23]dand some commercial methods, such as Sensititre YeastOne [24] and Etest [10,11,13]. In this study, we collected the MIC values (obtained with the Etest) for more than 18 000 Candida spp. and A. fumigatus SC isolates and calculated the Etest-based ECVs (n ¼ 48) using the ISM [7] at the 97.5% cut-off value to exclude isolates with extreme MICs. This analysis included the five Candida species responsible for more than 90% of invasive candidiasis and all the classes of antifungal agents rec- ommended in the guidelines [2,3] (except for C. glabrata and azoles), as well as less prevalent Candida species (C. lusitaniae, C. guilliermondii, C. kefyr), and A. fumigatus SC. Currently, only three other studies used the ISM and Etest data to calculate the ECVs (n ¼ 50) of some antifungal agents for six Candida spp. and five Aspergillus spp. [10,11,13]).. Table 2 Etest epidemiological cut-off values of amphotericin B and echinocandins in Candida spp. MIC mode (mg/L). Etest ECV (this study)a. Etest ECVs (Espinel-Ingroff)b. CLSI ECVsc. EUCAST ECVsd. CLSI BPs S�/R� e. EUCAST BPs S�/R >f. 11/12 11/12 10/11 10/12 9/11 9/11 9/11 6/11. 6062/6249 2272/2446 981/1137 787/866 534/603 353/380 170/189 131/164. 0.25 0.5 0.5 0.5 1 0.5 0.25 0.25. 1 2 2 2 4 2 1 1. 1 2 2 2 4 d d d. 2 2 2 2 2 d 2 2. 1 1 1 1 1 d d d. d d d d d d d d. 1 1 1 1 1 d d d. 5/5 5/5 3/4 5/5 3/3 11/11 11/11 11/11 11/11 5/11 7/11. ed. Used isolates/ total (n). 1593/1593 513/513 241/259 213/213 117/117. 0.004 0.016 2 0.016 0.03. 0.008 0.03 8* 0.03 0.12*. 0.016 0.03 8* 0.03 0.06. 0.12 0.12 8 0.12 0.25. 0.03 0.06 4 0.06 0.06. 0.25/1 0.12/0.5 2/8 0.25/1 0.25/1. 0.03/0.03 0.06/0.06 0.002/4 0.06/0.06 0.06/0.06. 3752/3752 1494/1494 839/839 504/504 259/402 236/254. 0.016 0.016 1 0.03 0.25 0.06. 0.03 0.03 4 0.06 0.5 0.25. 0.03 0.03 2 0.12 0.25 d. 0.03 0.03 4 0.06 0.25 0.12. 0.016 0.03 2 0.06 0.25 d. 0.25/1 0.06/0.25 2/8 0.25/1 0.25/1 d. 0.016/0.016 0.03/0.03 0.002/2 d d d. 5783/5783 2292/2303 1091/1109 787/798 565/568 418/446 149/175 120/152. 0.06 0.12 0.5 0.12 0.5 0.12 0.25 0.5. 0.25 0.5 2 0.5 1 0.25 1 2. 0.5 1 4 1 1 d d d. 0.12 0.12 1 0.12 0.25 0.03 1 2. d d d d d d d d. 0.25/1 0.12/0.5 2/8 0.25/1 0.25/1 d d 2/8. d d d d d d d d. ce. pt. Amphotericin B Candida albicans Candida glabrata SC Candida parapsilosis SC Candida tropicalis Candida krusei Candida kefyr Candida lusitaniae Candida guilliermondii Anidulafungin Candida albicans Candida glabrata SC Candida parapsilosis SC Candida tropicalis Candida krusei Micafungin Candida albicans Candida glabrata SC Candida parapsilosis SC Candida tropicalis Candida krusei Candida kefyr Caspofungin Candida albicans Candida glabrata SC Candida parapsilosis SC Candida tropicalis Candida krusei Candida kefyr Candida lusitaniae Candida guilliermondii. Contributing laboratories/total ( n). Ac. 12/12 11/12 10/12 10/12 10/12 8/10 7/10 5/9. Abbreviations: BP, breakpoint; ECV, epidemiological cut-off value; MIC mode, most frequent MIC in the distribution; R, resistant; S, susceptible; SC, species complex. *ECV obtained after data normalization (>50% of all data were from one laboratory). a ECVs calculated for the modelled population (�97.5%). b ECVs determined by Espinel-Ingroff et al. with the Etest method (�97.5%) [13]. c ECVs determined using the CLSI method in previous studies [9,15e17,19]. d ECVs determined using the EUCAST method [9,14,23]. e CLSI breakpoints [15,21]. f EUCAST breakpoints [6]..
(8) 6 Table 3 Etest epidemiological cut-off values for azoles and Candida spp. MIC mode (mg/L). EtestECVs (this study)a. EtestECVs (EspinelIngroff)b1. Etest ECVs (EUCAST determination)b2. CLSI ECVsc. EUCAST ECVsd. CLSI BPs S�/R� e. EUCAST BPs S�/R>f. 12/12 0/11 12/12 12/12 11/11 11/12 10/10 5/10. 9654/9654 0/2939 1458/1458 1148/1148 414/414 518/527 241/241 111/186. 0.25 d 0.5 1 >256 0.25 0.25 2. 1 d 2 4 d 1 1 4. d 64 4 4 d d d d. 1 32 2 2 128 d d d. 0.5 8 1 1 32 1 1 8. 1 32 2 2 128 d 16 16. 2/8 SDD�32/R � 64 2/8 2/8 d d d d. 2/4 0.002/32 2/4 2/4 d d d d. 12/12 0/11 12/12 12/12 10/12 9/11 8/11 5/10. 6020/6020 0/2304 1150/1150 886/886 575/673 357/374 168/189 115/163. 0.008 d 0.016 0.06 0.5 0.008 0.008 0.03. 0.03 d 0.12 0.25 1 0.03 0.03 0.12. 0.03 2 0.25 0.5 2 d d d. 0.12 1 0.12 0.12 1 d 0.06 0.25. 0.03 0.25 0.03 0.06 0.5 0.016 0.03 0.12. 0.12 1 0.12 0.12 1 d 0.06 0.25. 0.12/1 d 0.12/1 0.12/1 0.5/2 d d d. 0.06/0.25 d 0.12/0.25 0.12/0.25 d d d d. 6/7 0/7 4/6. 1005/1014 0/455 152/159. 0.016 d 0.03. 0.06 d 0.25*. 0.12 d 0.12. d d d. 0.06 2 0.12. 0.06 1 0.06. d d d. 0.06/0.06 d 0.06/0.06. rip. t. Used isolates/ total (n). M an us c. Fluconazole Candida albicans Candida glabrata SC Candida parapsilosis SC Candida tropicalis Candida krusei Candida kefyr Candida lusitaniae Candida guilliermondii Voriconazole Candida albicans Candida glabrata SC Candida parapsilosis SC Candida tropicalis Candida krusei Candida kefyr Candida lusitaniae Candida guilliermondii Posaconazole Candida albicans Candida glabrata SC Candida tropicalis. Contributing laboratories/ total (n). Abbreviations: BP, breakpoint; ECV, epidemiological cut-off value; MIC mode, most frequent MIC in the distribution; R, resistant; S, susceptible; SC, species complex. *ECV obtained after data normalization (>50% of all data were from one laboratory). a ECVs calculated for the modelled population (�97.5%). b1 ECVs determined by Espinel-Ingroff et al. with the Etest method (�97.5%) [11]. b2 Etest-based ECVs determined by the EUCAST organization [23]. c ECVs determined with the CLSI method in previous studies [9,11,12,17]. d ECVs determined with the EUCAST method [5,9,14,23]. e CLSI breakpoints [15,21]. f EUCAST breakpoints [6].. Ac. ce. pt. ed. For the Candida spp.eamphotericin B combinations, we deter- mined eight ECVs, of which three were new and five displayed the same values as those reported by Espinel-Ingroff et al. [13]. For the Candida spp.-echinocandin combinations, our Etest ECVs were identical (6/15 ECVs) or within one two-fold dilution (9/15 ECVs) compared with the previously published values. These non- significant discrepancies could be explained by the higher number of isolates analysed in our study (e.g. 5783 C. albicans isolates for caspofungin and 504 C. tropicalis isolates for micafungin versus 2537 and 140 isolates, respectively, in [13]). Espinel-lngroff et al. showed that the Etest ECVs for anidulafungin identified 92% of FKS mutants as non-WT isolates (versus 75% and 84% with the ECVs for caspofungin and micafungin, respectively) [13]. The authors concluded that the Etest ECVs for anidulafungin could represent a surrogate marker for echinocandin resistance screening in Candida spp., as also suggested by Pfaller et al. with the CLSI method [25]. In our study, the ECVs for caspofungin were most often lower (one two-fold dilution) than those reported by Espinel-Ingroff et al., which raised the question of selecting a robust surrogate marker for echinocandin resistance screening. To determine the most useful echinocandin ECV to identify non-WT isolates, it might be neces- sary to test isolates with characterized mutations. For the Candida spp.eazole combinations, among the Etest- based ECVs previously published [11], two were similar and six were within one two-fold dilution. Other Etest ECVs are available for azoles in the EUCAST website (https://mic.eucast.org/Eucast2/) [23], but they were determined using a small number of strains, sometimes <100 observations for some specieseantifungal agent combinations. There was no significant difference (i.e. more than one two-fold dilution) between our ECVs and the EUCAST ECVs, but for the C. albicansevoriconazole combination (ECV ¼ 0.03 mg/L in our study and 0.12 mg/L by EUCAST). However,. our ECV is identical to the one reported by Espinel-Ingroff et al., suggesting that this is a more robust value. We could not determine the Etest ECVs for C. glabrata SC and azoles because of the aberrant MIC distribution (double peak). Indeed, there was a significant number of strains with high MICs (>256 mg/L) due to the appearance of ‘macro-col- onies’ in the inhibition ellipse when MICs were read at 48 h. Some authors have found lower rates of agreement for C. glabrata and azoles when comparing the Etest and the CLSI method (higher MICs with the Etest) [26,27]. The incubation time seems to influence the results. Indeed, a better agreement between these methods is observed.
(9) Ac. ce. pt. ed. M an us c. rip. t. at 24 h of growth and reading. In their latest study to determine the ECVs for C. glabrata SC and azoles,7 Espinel-Ingroff et al. read the Etest MICs between 24 h and 48 h, depending on the growth [11]. This may explain why they did not observe double peaks and could, therefore, estimate the ECVs. In our study, we could determine the Etest ECVs for antifungal agents used as first-line and also salvage therapy of the most prevalent species of invasive aspergillosis (A. fumigatus SC). In contrast to our study, Espinel-Ingroff et al. observed a significant heterogeneity in the minimum effective concentration mode for Aspergillus spp. and caspofungin, and could not determine the Etest ECVs [13]. The new identification methods (especially matrix- assisted laser desorption/ionization time-of-flight) that can also identify cryptic species will allow determination of the ECVs for species within a complex. This is important because the suscepti- bility to an antifungal drug is not the same for all species within a complexdfor example, Aspergillus lentulus shows higher MICs to amphotericin B) [28]..
(10) Table 4 Etest epidemiological cut-off values for the Aspergillus fumigatus species complex Used isolates/ total (n). MIC mode (mg/L). EtestECVs (this study)a. Etest ECVs (Espinel-Ingroff)b. CLSI ECVsc. EUCAST ECVsd. CLSI BPs S�/R�. EUCAST BPs S�/R>e. 7/10 8/10 7/8 9/10 5/9 5/8. 1526/1626 961/1035 989/1064 1027/1039 806/892 361/372. 0.12 0.12 0.5 0.5 0.03 0.008. 0.5 0.25 2 2 0.12* 0.016*. 0.5 0.25 2 2 d d. 1 0.25 1 2 0.5 d. 1 0.25 1 1 d d. d d d d d d. 1/2 0.125/0.25 1/2 1/2 d d. t. Voriconazole Posaconazole Itraconazole Amphotericin B Caspofungin Micafungin. Contributing laboratories/total (n). M an us c. rip. Abbreviations: BP, breakpoint; ECV, epidemiological cut-off value; MIC mode, most frequent MIC in the distribution; R, resistant; S, susceptible; SC, species complex *ECV obtained after data normalization (>50% of data were from one laboratory). a ECVs calculated for the modelled population (�97.5%). b ECVs based on the Etest method determined by Espinel-Ingroff et al. (�97.5%) [10,11,13]. c ECVs determined with the CLSI method in previous studies [10,13]. d ECVs determined with the EUCAST method [5,9,10,23]. e EUCAST breakpoints [6].. ce. pt. ed. All the previously published ECVs were calculated by only one team. Our multi-laboratory study allowed consolidation of these previous ECV data and validation of our results. Specifically, 17/32 of our ECVs are identical to previous ones, and 15/32 show just one two-fold dilution [10,11,13], a difference that we consider not sig- nificant (our definition is more stringent than the essential agree- ment, which considers two log2 dilutions). These not significant differences can be explained by the higher number of laboratories and isolates included in our study for the ECV calculations. How- ever, ideally only one ECV should be proposed for each specieseantifungal combination. For that, our data and the previ- ous data should be combined and analysed again to reach a consensus. Comparison of our Etest ECVs with the ECVs obtained with the reference methods indicated that they were identical to or within one two-fold dilution of the CLSI ECVs in 76.1% of cases [9,12,15e17,19] and to the EUCAST ECVs in 81.2% of cases [9,10,14,22]. Discrepancies among ECVs obtained with different methods were previously reported [13,24], emphasizing the importance of using ECVs specific for the in vitro method used to test the antifungal susceptibility. Moreover, for each specieseantifungal combination, it is possible to know whether the MIC distributions obtained with the Etest are close to the distri- butions obtained with the CLSI and EUCAST methods. For example, for C. lusitaniae and fluconazole, the Etest MIC distributions were identical to those obtained with the CLSI but not the EUCAST method (difference of four log2 dilutions). This can be useful for using the available breakpoints. As Etest is the most widely used commercial method in French laboratories, the goal of this study was to calculate specific ECVs for this method. Inter-method variability and comparison of the Etest method with a reference method were previously performed with good essential agreement [26,29,30]. It is now important to have data on Etest MIC distributions for MIC interpretation in routine clinical practice and for the detection of non-WT isolates. When the MIC value is higher than the ECV, the search for possible gene mutations to that antifungal agent should be recommended.. Ac. Conclusions. This study identified 48 ECVs specific for the Etest method to facilitate MIC interpretation. They should not be used instead of breakpoints, but they may be useful to identify non-WT isolates with potential resistance to antifungal agents and to suggest that an isolate may not respond as expected to the standard treatment. ECVs have a place in the surveillance and monitoring of the emergence of drug resistance. It will be interesting to prospectively collect and pool MIC data to confirm the calculated Etest ECVs and to determine other ECVs, especially for rare species. Testing isolates with acquired resistance mutations characterized by molecular methods will also provide a way to verify these ECVs..
(11) Transparency declaration The authors declare that there are no conflicts of interest rele- vant to this article.. Acknowledgements. rip. t. The authors thank I. Accoceberry, N. ait Ammar, N. Argy, A. Berry, C. Bonnal, P. Chauvin, N. Coron, J. Fillaux, F. Foulet, F. Gabriel, X. Iriart, P. Millet, P. Rispail, G. Roux and A. Valentin for their partici- pation in the study and thank all the laboratory staff for their technical assistance.. M an us c. References. Ac. ce. pt. ed. [1] Dromer F, Bretagne S, Lortholary O. Rapport annuel d’activite' - centre National de re'fe'rence Mycoses Invasives et Antifongiques. Paris: Institut Pasteur; 2016. [2] Ullmann AJ, Aguado JM, Arikan-Akdagli S, Denning DW, Groll AH, Lagrou K, et al. Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline. Clin Microbiol Infect 2018;24: e1e38. [3] Cornely OA, Bassetti M, Calandra T, Garbino J, Kullberg BJ, Lortholary O, et al. ESCMID Guideline for the diagnosis and management of Candida diseases 2012: non-neutropenic adult patients. Clin Microbiol Infect 2012;18:19e37. [4] Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts. Fourth informational sup- plement M27-S4. Wayne, PA: CLSI; 2012. [5] Arendrup MC, Cuenca-Estrella M, Lass-Flo€rl C, Hope WW. Breakpoints for antifungal agents: an update from EUCAST focussing on echinocandins against Candida spp. and triazoles against Aspergillus spp. Drug Resist Updat 2013;16: 81e95. [6] European Committee on Antimicrobial Susceptibility Testing EUCAST. Antifungal_breakpoints_v_9.0_180212. EUCAST; 2018. [7] Turnidge J, Kahlmeter G, Kronvall G. Statistical characterisation of bacterial wild-type MIC value distributions and the determination of epidemiological cut-off values. Clin Microbiol Infect 2006;12:418e25. [8] Lockhart SR, Ghannoum MA, Alexander BD. Establishment and use of epide- miological cutoff values for molds and yeasts by use of the Clinical and Lab- oratory Standards Institute M57 Standard. J Clin Microbiol 2017;55:1262e8. [9] Espinel-Ingroff A, Turnidge J. The role of epidemiological cutoff values (ECVs/ ECOFFs) in antifungal susceptibility testing and interpretation for uncommon yeasts and moulds. Rev Iberoam Micol 2016;33:63e75. [10] Espinel-Ingroff A, Turnidge J, Alastruey-Izquierdo A, Dannaoui E, GarciaEffron G, Guinea J, et al. Posaconazole MIC distributions for Aspergillus fumi- gatus species complex by four methods: impact of cyp51A mutations on estimation of epidemiological cutoff values. Antimicrob Agents Chemother 2018;62:e01916e7. [11] Espinel-Ingroff A, Turnidge J, Alastruey-Izquierdo A, Botterel F, Canton E, Castro C, et al. Method-dependent epidemiological cutoff values (ECVs) for detection of triazole resistance in Candida and Aspergillus species for the SYO colorimetric broth and Etest agar diffusion methods. Antimicrob Agents Chemother 2018;63. e01651-18. [12] Espinel-Ingroff A, Pfaller MA, Bustamante B, Canton E, Fothergill A, Fuller J, et al. Multilaboratory study of epidemiological cutoff values for detection of.
(12) 1. Ac. ce. pt. ed. M an us c. rip. t. resistance in eight Candida species to fluconazole, posaconazole, and vor- iconazole. Antimicrob Agents Chemother 2014;58:2006e12. [13] Espinel-Ingroff A, Arendrup M, Canto'n E, Cordoba S, Dannaoui E, GarcíaRodríguez J, et al. Multicenter study of method-dependent epidemiological cutoff values for detection of resistance in Candida spp. and Aspergillus spp. to Amphotericin B and echinocandins for the Etest agar diffusion method. Anti- microb Agents Chemother 2016;61. e01792-16. [14] Meletiadis J, Curfs-Breuker I, Meis JF, Mouton JW. In vitro antifungal suscep- tibility testing of Candida isolates with the EUCAST methodology, a new method for ECOFF determination. Antimicrob Agents Chemother 2017;61. e02372-16. [15] Pfaller MA, Diekema DJ. Progress in antifungal susceptibility testing of Candida spp. by use of Clinical and Laboratory Standards Institute broth microdilution methods, 2010 to 2012. J Clin Microbiol 2012;50:2846e56. [16] Pfaller MA, Espinel-Ingroff A, Bustamante B, Canton E, Diekema DJ, Fothergill A, et al. Multicenter study of anidulafungin and micafungin MIC distributions and epidemiological cutoff values for eight Candida species and the CLSI M27-A3 broth microdilution method. Antimicrob Agents Chemother 2014;58:916e22. [17] Clinical and Laboratory Standards Institute. Epidemiological cutoff values for antifungal susceptibility testing M59. Wayne, PA: CLSI; 2016. [18] Espinel-Ingroff A, Fothergill A, Fuller J, Johnson E, Pelaez T, Turnidge J. Wildtype MIC distributions and epidemiological cutoff values for caspofungin and Aspergillus spp. for the CLSI Broth Microdilution Method (M38-A2 Document). Antimicrob Agents Chemother 2011;55:2855e9. [19] Pfaller MA, Espinel-Ingroff A, Canton E, Castanheira M, Cuenca-Estrella M, Diekema DJ, et al. Wild-type MIC distributions and epidemiological cutoff values for amphotericin B, flucytosine, and itraconazole and Candida spp. as determined by CLSI broth microdilution. J Clin Microbiol 2012;50:2040e6. [20] biome'rieux SA. Etest antifungal susceptibility testing for in vitro diagnostic use package insert. Marcy l'E'toile: bioMe'rieux; 2013. [21] biome'rieux SA. Summary of etest. Performance interpretative criteria and quality control ranges. Marcy l'E'toile: bioMe'rieux; 2018..
(13) [22] Howard SJ, Lass-Flo€rl C, Cuenca-Estrella M, Gomez-Lopez A, Arendrup MC. Determination of isavuconazole susceptibility of Aspergillus and Candida species by the EUCAST method. Antimicrob Agents Chemother 2013;57: 5426e31.. Ac. ce. pt. ed. M an us c. rip. t. [23] European Committee on Antimicrobial Susceptibility Testing EUCAST. EUCAST Antimicrobial wild type distributions of microorganisms. Available at: https:// MicEucastOrg/Eucast2/2019. https://mic.eucast.org/Eucast2/(accessed 11 December 2018). [24] Morris AJ, Rogers K, McKinney WP, Roberts SA, Freeman JT. Antifungal sus- ceptibility testing results of New Zealand yeast isolates, 2001e2015: impact of recent CLSI breakpoints and epidemiological cut-off values for Candida and other yeast species. J Glob Antimicrob Resist 2018;14:72e7. [25] Pfaller MA, Diekema DJ, Jones RN, Castanheira M. Use of anidulafungin as a surrogate marker to predict susceptibility and resistance to caspofungin among 4,290 clinical isolates of Candida by using CLSI methods and inter- pretive criteria. J Clin Microbiol 2014;52:3223e9. [26] Alexander BD, Byrne TC, Smith KL, Hanson KE, Anstrom KJ, Perfect JR, et al. Comparative evaluation of etest and Sensititre YeastOne panels against the clinical and laboratory standards Institute M27-A2 reference broth micro- dilution method for testing Candida susceptibility to seven antifungal agents. J Clin Microbiol 2007;45:698e706. [27] Metin DY, Hilmioglu-Polat S, Samlioglu P, Doganay-Oflazoglu B, Inci R, Tumbay E. Evaluation of antifungal susceptibility testing with microdilution and Etest methods of Candida blood isolates. Mycopathologia 2011;172: 187e99. [28] Alastruey-Izquierdo A, Alcazar-Fuoli L, Cuenca-Estrella M. Antifungal sus- ceptibility profile of cryptic species of Aspergillus. Mycopathologia 2014;178: 427e33. [29] Aigner M, Erbeznik T, Gschwentner M, Lass-Flo€rl C. Etest and Sensititre YeastOne susceptibility testing of echinocandins against Candida species from a single center in Austria. Antimicrob Agents Chemother 2017;61:e00512e7. [30] Dannaoui E, Paugam A, Develoux M, Chochillon C, Matheron J, Datry A, et al. Comparison of antifungal MICs for yeasts obtained using the EUCAST method in a reference laboratory and the Etest in nine different hospital laboratories. Clin Microbiol Infect 2010;16:863e9..
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